This invention relates to gene therapy and drug delivery. More particularly, the invention relates to compositions and methods for use, and making thereof, for delivering nucleic acids as gene therapy applications or other non-soluble bioactive molecules such as protein, peptides or small non-soluble drugs.
Biodegradable polymers are gaining attention as drug delivery systems. R. Langer, New Methods of Drug delivery, 249 Science 1527-1533 (1990); B. Jeong et al., Biodegradable Block Copolymers as Injectable Drug-delivery Systems, 388 Nature 860-862 (1997). Since genes are now considered pharmaceutical agents for treating many types of diseases, and gene therapy is becoming widely used as demonstrated by many clinical trials, M. A. Kay et al., Gene Therapy, 94 Proc. Nat'l Acad. Sci. USA 1744-12746 (1997); C. Bordignon et al., Gene Therapy in Peripheral Blood Lymphocytes and Bone Marrow for ADA-immunodeficient Patients, 270 Science 470-475 (1995), there is an urgent need for a safe and efficient gene carrier. Genes are very attractive candidates for therapeutic use in a variety of disease states due to their ability to produce bioactive proteins using the biosynthetic machinery provided by host cells. A major technical impediment to gene transfer is the lack of an ideal gene delivery system. There are many established protocols for transferring genes into cells, including calcium phosphate precipitation, electroporation, particle bombardment, liposomal delivery, viral-vector delivery, and receptor-mediated gene delivery. A.V.Kavanov, Self-assembling Complexes for Gene delivery, p.L.Felgner & L.W.Seymour, J.Wiley & Sons (1998); P.L.Chang, Somatic Gene Therapy, CRC Press (1995).
Transfection methods using retroviral or adenoviral vectors have been investigated. Retroviral vectors, in particular, have been used successfully for introducing exogenous genes into the genomes of actively dividing cells such that stable transformants are obtained. D. G. Miller et al., Gene Transfer by Retrovirus Vectors Occurs Only in Cells that are Actively Replicating at the Time of Infection. 10 Mol. Cell Biol. 4239-4242 (1990). Viral vector systems often, in case of complementation of defective vectors by inserting genes into `helper` cell lines, generate a transducing infectious agent. In addition, it is well known that the host immune response to adenoviruses limits their use as a transfer facilitating agent to a single administration. To address this limitation, fusion peptides of the influenza virus hemagglutinin have been employed to replace adenoviruses as endosomal lytic agents, but have met with limited success. S. Gottschalk et al., A Novel DNA-Peptide Complex for Efficient Gene Transfer and Expression in Mammalian Cells, 3 Gene Ther. 448-457 (1996). However, despite their high transfection efficiency in vitro, inserting genes into the host cell's genome in vivo depends on the viral infection pathway. Application of the viral infection pathway for human gene therapy introduces serious concerns about endogenous virus recombination, oncogenic effects, and inflammatory or immunologic reactions. G Ross et al., Gene Therapy in the United States: A Five-Year Status Report. 7 Hum. Gene Ther., 1781-1790 (1996). Because of these concerns the use of viral vectors for human gene therapy has been extremely limited.
As compared to viral gene carriers, there are several advantages to the use of non-viral based gene therapies, including their relative safety and low cost of manufacture. Non-viral gene delivery systems such as cationic liposomes or synthetic gene carriers, e.g.poly-L-lysine (PLL), are being widely sought as alternatives and investigated intensively to circumvent some problems encountered in viral vectors. K. A. Mislick et al., Transfection of Folate-polylysine DNA Complexes: Evidence for Lysosomal Delivery, 6 Bioconjugate Chem. 512-515 (1995); J.O. Radler et al., Structure of DNA-cationic Liposome Complexes: DNA Intercalation in Multilamellar Membranes in Distinct Interhelical Packing Regimes, 275 Science 810-814 (1997); J. Cheng et al., Effect of Size and Serum Proteins on Transfection Efficiency of Poly((2-dimethylamino)ethyl methacrylate)-plasmid nanoparticles, 13 Pharm. Res. 1038-1042 (1996). There are several polymeric materials currently being investigated for use as gene carriers, of which poly-L-lysine (PLL)is the most popular, but few of them are biodegradable. Biodegradable polymers, such as polylactic/glycolic acid(negatively charged), and polylactide/glycolide (neutral) have been used as gene carriers in the form of non-soluble particulates. Amarucyama et al, Nanoparticle DNA Carrier with PLL Grafted Polysallanide Copolymer and Polylactic Acid, 8 Bioconjugate, 735-739(1997). In general, polycationinic polymers are known to be toxic and the PLL backbone is barely degraded under physiological conditions. It will remain in cells and tissue which cause undesirably high toxicity. A.Segouras & R.Dunlan, Methods for Evaluation of Biocompatibility of Synthetic Polymers, 1 J.Mater.Sci in Medicine, 61-68(1990).
In view of the foregoing it will be appreciated that providing a soluble and biodegradable gene carrier, meaning that the polymer gene carrier can break down or degrade within body to non-toxic components after the genes have been delivered, that is non viral, safe and effective would be a significant advancement in the art.